The hyperthermophilic and radioresistant euryarchaeon Thermococcus gammatolerans encodes a putative 3-methlyadenine DNA glycosylase II (Tg-AlkA). Herein, we report biochemical characterization and catalytic mechanism of Tg-AlkA. The recombinant Tg-AlkA can excise hypoxanthine (Hx) and 1-methlyadenine (1-meA) from dsDNA with varied efficiencies at high temperature. Notably, Tg-AlkA is a bi-functional glycosylase, which is sharply distinct from all the reported AlkAs. Biochemical data show that the optimal temperature and pH of Tg-AlkA for removing Hx from dsDNA are ca.70 °C and ca.7.0–8.0, respectively. Furthermore, the Tg-AlkA activity is independent of a divalent metal ion, and Mg²⁺ stimulates the Tg-AlkA activity whereas other divalent ions inhibit the enzyme activity with varied degrees. Mutational studies show that the Tg-AlkA W204A and D223A mutants abolish completely the excision activity, thereby suggesting that residues W204 and D223 are involved in catalysis. Surprisingly, the mutations of W204, D223, Y139 and W256 to alanine in Tg-AlkA lead to the increased affinity for binding DNA substrate with varied degrees, suggesting that these residues are flexible for conformational change of the enzyme. Therefore, Tg-AlkA is a novel AlkA that can remove Hx and 1-meA from dsDNA, thus providing insights into repair of deaminated and alkylated bases in DNA from hyperthermophilic Thermococcus.

超嗜热和抗辐射的广古细菌Thermococcus gammatolerans编码假定的 3-甲基腺嘌呤 DNA 糖基化酶 II (Tg-AlkA)。在此，我们报告了 Tg-AlkA 的生化表征和催化机制。重组 Tg-AlkA 可以在高温下以不同的效率从 dsDNA 中切除次黄嘌呤 (Hx) 和 1-甲基腺嘌呤 (1-meA)。值得注意的是，Tg-AlkA 是一种双功能糖基化酶，与所有报道的 AlkA 截然不同。生化数据表明，Tg-AlkA 从 dsDNA 中去除 Hx 的最佳温度和 pH 值分别为约 70 °C 和约 7.0-8.0。此外，Tg-AlkA 活性不依赖于二价金属离子，Mg ²⁺刺激 Tg-AlkA 活性，而其他二价离子不同程度地抑制酶活性。突变研究表明，Tg-AlkA W204A 和 D223A 突变体完全消除了切除活性，从而表明残基 W204 和 D223 参与催化。令人惊讶的是，Tg-AlkA 中 W204、D223、Y139 和 W256 向丙氨酸的突变导致不同程度地结合 DNA 底物的亲和力增加，表明这些残基对于酶的构象变化是灵活的。因此，TG-Alka的是一种新型的ALKA可从双链DNA除去HX 1和-MEA，从而提供见解的从超嗜热DNA中脱氨基和烷基化的碱基修复嗜热。